2.1. Control of Bleeding
Mammals control bleeding by producing platelets, which are activated with agents that are produced or released at the site of a wound. Activation is necessary for the platelets to aggregate into clumps or clots.
The activation of platelets is a complicated process, which includes producing or exposing receptors for the plasma protein fibrinogen on the platelet surface. Fibrinogen has multiple binding sites, and binds two or more platelets simultaneously, initiating the aggregation. A platelet receptor that is present on the surface of platelets and becomes exposed during the activation process is GPIIb/IIIa. Patients with low platelet counts often require transfusions of platelets in order to control bleeding.
In 1910, Duke provided data suggesting that transfusion of whole blood containing platelets could arrest hemorrhage due to thrombocytopenia (Duke, 1910, J. Am. Med. Assoc. 60:1185-1192). It was not, however, until the 1950's that unequivocal data on the efficacy of platelet transfusions were obtained in animals made thrombocytopenic by treatment with total body irradiation (Cronkite et al., 1959, in Progress in Hematology, vol. 2, Tocantins, ed, Grune and Stratton, N.Y., pp. 239-257). The difficulties in platelet procurement and storage led investigators to seek alternatives to fresh platelets soon thereafter. Studies performed with lyophilized platelets and disintegrated platelets indicated that these products failed to arrest bleeding (Cronkite et al., supra; Jackson et al., 1959, J. Clin. Invest. 38:1689; Hjort et al., 1959, Proc. Soc. Exp. Biol. Med. 102:31-35). When phospholipids were found capable of substituting for platelets in accelerating coagulation reactions, the cephalin fraction of soy bean phosphatides was investigated as a platelet substitute in thrombocytopenic children (Shulman et al., 1959, Ann. N.Y. Acad. Sci. 75:195, Abstr.). Although a preliminary report suggested clinical improvement in some patients (Schulman et al., supra) animal studies did not identify a benefit and this approach was eventually abandoned (Kahn, et al., 1985 Blood 66:1-12, Abstr.). Cryopreservation of autologous platelets has been successfully utilized in patients with problems related to alloimmunization and isoimmunization (Schiffer et al., N. Engl. J. Med. 299:7-12), but the platelet yield is less than with fresh platelets, and the technique is limited by the need for extra processing and the availability of storage space (Murphy, 1991, In Principles of Transfusion Medicine, Williams & Wilkins, Baltimore, pp. 205-213).
Improved understanding of platelet physiology has led to additional approaches to obtain a platelet substitute. Several investigators have been able to introduce platelet glycoproteins into liposomes for in vitro experiments (Parise and Phillips, 1985, J. Biol. Chem. 260:1750-1756; Baldassare et al., 1988, J. Clin. Invest. 75:35-39; Rybak, 1986, Thromb. Haemostas. 55:240-245). More recently, Ryback and Renzulli incorporated a deoxycholate extract of platelet membranes containing 15 proteins, including GPIb, GPIIb/IIIa, and GPIV, into small (50-200 nm) unilamellar liposomes prepared from either sphingomyelin:phosphatidylcholine:monosialyloganglioside or egg phosphatide (Blood Suppl. 1:473a, abstr). Intra-arterial injections of both preparations decreased bleeding in thrombocytopenic rats to the same extent as human platelets did, but neither produced complete normalization of the bleeding time. Interestingly, liposomes containing GPIIb/IIIa alone were ineffective (Rybak and Renzulli, 1990, Blood Suppl. 1:473a, Abstr.). This approach may provide important mechanistic information but as a therapeutic intervention it potentially suffers from the generic problems of liposomes, including the possibility of short in vivo survival and potential blockade of the reticuloendothelial system (Kahn et al., 1985, Blood 66:1-12, Abstr.). Moreover, since platelets remain the starting material, problems of platelet procurement and the risks of transmitting infectious diseases may not be eliminated. Finally, if whole platelet extracts are required, immunogenicity may limit the opportunity for repeat therapy because platelets have class I HLA antigens (McFarland and Aster, 1991, In Principles of Transfusion Medicine, Williams & Wilkins, Baltimore, pp. 193-204), and some platelet glycoproteins are polymorphic (Lopez and Ludwig, 1991, Clin. Res. 39:327 a.s.).
Agam and Livne took an approach based on their observations that passive, fixed platelets coated with fibrinogen could function to augment platelet aggregation of native, fresh platelets (Agam and Livne, 1983, Blood 61:186; Agam and Livne, 1984, Thromb. Haemostas. 51:145-149; Agam and Livne, 1988, Thromb. Haemostas. 59:504-506). They concluded that the activated platelets had to undergo the release reaction and expose thrombospondin on their surface in order for the interactions to occur, with the final interaction between the fibrinogen on the fixed platelets and the thrombospondin on the activated platelets (Agam and Livne, 1983, Blood 61:186; Agam and Livne, 1984, Thromb. Haemostas. 51:145-149; Agam and Livne, 1988, Thromb. Haemostas. 59:504-506). This suggests that the fixation process alters the fibrinogen so that it cannot interact directly with GPIIb/IIIa, but leaves intact portions of the fibrinogen molecule that can interact with thrombospondin. This approach involves a significant limitation in that it relies on the purification of fibrinogen from plasma, and thus has the risk of transmitting blood-borne disease. Moreover, formaldehyde is a cytotoxic agent that may have carcinogenic potential (Feron et al., 1991, Mutation Res. 259:363-385) and so it may not be the most desirable crosslinking reagent for in vivo use.
Until the present invention, platelet transfusion was the only effective therapy for the prevention and treatment of hemorrhage due to thrombocytopenia (Heyman et al., 1991, in Principles of Transfusion Medicine, William & Wilkens, Baltimore, pp. 223-231). The number of units of platelets transfused each year in the United States has grown rapidly since the widespread introduction of platelet transfusion therapy in the 1960's; in fact, just between 1980 and 1987 the number nearly doubled, reaching in excess of 6 million units per year (Surgenor et al., 1990, N. Engl. J. Med. 322:1646-1651). Despite its enormous success, platelet transfusion therapy has a number of very serious limitations and drawbacks: 1) supplies are often limited due to difficulties in procurement and the relatively short shelf life (5-7 days) (Murphy, 1991, in Principles of Transfusion Medicine, Williams & Wilkens, Baltimore, pp. 205-213); 2) there is a risk of transmitting blood-borne pathogens such as the viruses that produce hepatitis and AIDS, especially since multiple units are usually administered with each transfusion (Heyman et al., supra); 3) febrile reactions, presumably due to white blood cell contaminants, are common in patients receiving repeated transfusions (Snyder et al., 1991, in Principles of Transfusion Medicine, Williams & Wilkens, Baltimore, pp. 641-648); 4) alloimmunization results in patients becoming refractory to random donor platelets, necessitating a switch to single donor platelets matched for HLA antigens, and even HLA matched platelet transfusions are not universally successful (Heyman, et al., supra).
The interaction between fibrinogen and platelets has been the subject of prior investigations. For example, platelets interact with fibrinogen-coated polyacrylonitrile beads via a mechanism involving fibrinogen receptors on platelet surfaces (see the paragraph bridging pages 177 and 178 in Coller et al., 1980, Blood, 55:169-178). Agam and Livne (1983, Blood 61:186-191) disclosed that fixed platelets to which fibrinogen had been bound participated in the aggregation of activated platelets, by selective reaction with activated platelets. Ruoslahti et al., U.S. Pat. No. 4,792,525 suggested that the ability of proteins such as fibrinogen to interact with cells is associated with the amino acid sequence Arg-Gly-Asp-Ser within the fibrinogen structure. The Ruoslahti et al. patent further discloses that a tetrapeptide consisting of the sequence Arg-Gly-Asp-Ser, when properly immobilized on a substrate, has the property of causing cell attachment to the substrate. The tetrapeptide could be extended with additional amino acids at either end, and the possibility of very limited substitution for the amino acids constituting the tetrapeptide was suggested. A practical application envisioned by Ruoslahti et al. was platelet aggregation.
Despite the known role of platelets in controlling bleeding and of the interaction between fibrinogen and platelets, there are no current procedures for controlling bleeding in thrombocytopenic patients other than platelet transfusions, with all the disadvantages of such transfusions, as discussed above.
The availability of an abundant and safe alternative to human platelets would, therefore, be of considerable benefit. It is vital, however, that such an alternative retain the platelet's specificity for forming thrombi at sites of vascular injury, to be certain that indiscriminate thrombus formation does not occur. Prior to the instant invention, no abundant safe alternative to human platelets has been found.
Alternative procedures are needed in order to reduce the large amount of blood necessary to obtain sufficient platelets. The ideal procedure would utilize a patient's own blood cells in order to reduce the possibility of blood borne disease.
In addition, the precise delivery of radiolabeled molecules, diagnostic, and therapeutic agents to specific target tissues is an important laboratory and clinical problem.
Accordingly, one objective of the present invention is to solve the problems of obtaining cells from a small amount of blood, particularly autologous blood, that can be used to deliver precisely agents to specific target issues.
Another objective is to provide compositions of matter that are able to bind selectively to activated platelets but not to unactivated platelets in vivo. Activation refers to the process by which platelets become more susceptible to aggregation. The process by which platelets become activated is poorly understood, especially, in vivo. It appears that the activation process is induced by a number of agonists, such as ADP, epinephrine, collagen, thrombin and thromboxane A2. Indiscriminate binding of an agent to both activated and unactivated platelets exposes the patient to the risk of thrombosis (blood clots) that can lead to the death of tissues in vital organs, including the heart and brain.